Project 2: Multiple Antigen-Engineered DC Immunization and IFNa Boost for Metastatic Melanoma This application will build on progress made in our previous tumor antigen targeting trials to test a new genetically engineered dendritic cell-based vaccine regimen designed to more potently activate CDS and CD4 T cells specific to multiple melanoma antigens. We will couple this vaccine trial with thorough immunological monitoring to study T cell responses to the vaccine and the importance of determinant spreading for clinical response. We hypothesize that vaccination with multiple full length tumor antigens will activate a broad range of CD4 and CDS T cells, and that in the subset of patients who further activate and diversify their T cell response to include other antigens expressed by their tumor (or undergo determinant spreading), objective clinical response will be observed. We also hypothesize that systemic IFNa delivered after the vaccine will boost the vaccine-specific T cell responses. A. Specific Aims: A.1. Conduct an Antigen-Engineered DC Trial with an IFNa Boost. We will treat 30 subjects with antigen engineered DC and randomize half to receive an IFNa boost. A.2.Assess the Biology of the CDS and CD4 Immune Responses to Immunizing Antigens MART-1, Tyrosinase and MAGE-A6. We will follow CDS and CD4 T cell responses to the three immunizing antigens, as well as the adenovirus vector. We will also investigate TIL responses to the immunizing antigens and tumor antigen expression in accessible tumor deposits. A.3. Assess Determinant Spreading. We will also follow the CDS and CD4 T cell responses to defined melanoma-associated antigens not included in the vaccine but commonly expressed by tumors (including gplOO) and uncharacterized antigens expressed by autologous tumor to determine the importance of determinant spreading to objective clinical response.